Cyanobacterial PPP Family Protein Phosphatases Possess Multifunctional Capabilities and Are Resistant to Microcystin-LR

Abstract

The structural gene for a putative PPP family proteinserine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine-and phosphotyrosine-containing proteins and 3-phosphohistidine-and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.Strains of the cyanobacterium Microcystis aeruginosa sp. synthesize the cyclic heptapeptide microcystin-LR, a potent toxin toward humans and other animals (1, 2). In eukaryotes, the immediate targets of microcystin-LR are the structurally homologous catalytic subunits of the protein-serine/threonine phosphatases PP2A and PP1 1 (3), to which it binds with nanomolar or near nanomolar affinity, respectively (4). Sensitivity to these and other toxins, such as okadaic acid, is so highly conserved among eukaryotic PP1 and PP2A that it serves as a criterion for the identification of these enzymes in cell extracts (5). Moderate sensitivity to microcystin-LR extends to homologs recently identified in members of the Archaea such as Methanosarcina thermophila TM-1 (6, 7) and Pyrodictium abyssi TAG11 (8).Recently, the presence of open reading frames potentially encoding PP1/2A-like, also known as the PPP family (9), protein phosphatases has been reported in two strains of M. aeruginosa, the microcystin-producing strain M. aeruginosa PCC 7820 and the non-producing strain M. aeruginosa UTEX 2063 (10). These open reading frames have been designated pp1-cyano1 and pp1-cyano2, respectively. Given the observation that microcystins accumulate within the interior of toxin-producing cyanobacteria (11), and the near absolute conservation of sensitivity to these compounds by members of the PP1/2A superfamily (5), how do microcystin-producing cyanobacteria protect themselves from the action of endogenous toxins against their presumptive PP1/ 2A-like protein phosphatases? It ...

Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806

“…More than 60 isoforms of this heptapeptide are currently known, sharing the structure cyclo(--Ala--X--MeAsp--Z-Adda--Glu-Mdha), where X and Z represent variable -amino acids. Microcystins are specific inhibitors of the eukaryotic protein phosphatases 1 and 2A (Runnegar et al, 1993), but not of a similar cyanobacterial protein phosphatase (Shi et al, 1999). They are synthesized by various cyanobacterial freshwater species belonging to the genera Microcystis, Anabaena, Oscillatoria and Nostoc (Chorus & Bartram, 1999).…”

The PPP-Family Protein Phosphatases PrpA and PrpB of Salmonella enterica Serovar Typhimurium Possess Distinct Biochemical Properties

“…Unfortunately, divalent cation selectivity cannot be compared since this was not determined for the E. coli enzymes. The effects of several phosphatase inhibitors on ST-PrpA and ST-PrpB were generally similar not only to each other but also to the effects of the same inhibitors on the homologous phosphatases PP1-cyano1 from Microcystis aeruginosa PCC 7820 and PP1-cyano2 from M. aeruginosa UTEX 2063 (16). In contrast, although fluoride ion could completely block phosphatase activity in the serovar Typhimurium and M. aeruginosa enzymes, it had little effect on the two E. coli phosphatases.…”

“…The effect of protein phosphatase and other phosphomonoesterase inhibitors (11,14,16) on the activity of ST-PrpA and ST-PrpB was also examined (data not shown). At the concentrations tested, pyrophosphate, the chelating agent EDTA, the nonspecific phosphatase inhibitor NaF, and the tyrosine phosphatase inhibitor vanadate inhibited Ͼ90% of activity from either enzyme with […”

“…arch1 from the archaeon Sulfolobus solfataricus and PP1-cyano1 and PP1-cyano2 from Microcystis sp. (7,12,16 PrpA and PrpB of E. coli have also been purified and partially characterized (13). Unfortunately, divalent cation selectivity cannot be compared since this was not determined for the E. coli enzymes.…”

“…Homologs of PPP phosphatases can be discerned in many bacterial and archaeal genomes (for reviews, see references 8 and 17). All bacterial PPP phosphatases characterized to date require Mn 2ϩ for maximal activation (7,10,(12)(13)(14)(15)(16)(18)(19)(20). To investigate the role of Mn 2ϩ further, we have cloned, purified, and characterized the two PPP phosphatase homologs evident in the extant genome of serovar Typhimurium.…”